PROJECT SUMMARY Nuclease-based somatic genome editing, including approaches that use CRISPR/Cas9 and DNA Base Editor (BE), is a transformative technology that has the potential to cure many human diseases. However, to translate genome editing into widespread clinical use, there is an unmet need for safer and more effective technologies to deliver genome editing machinery into disease-relevant somatic cells and tissues in vivo. Although Adeno- Associated Viral (AAV) vectors are capable of delivering CRISPR/Cas9 systems in vivo with high editing efficiency, they have limited packaging capacity, lack the specificity in targeting cells/tissues, and can induce genotoxicity and immune responses due to persistent expression of Cas9. Further, most nonviral methods for in vivo delivery of CRISPR/Cas9 using systemic administration remain ineffective. To address these challenges, we have developed the Velcro AAV platform ? AAV vectors with Leucine Zippers (LZ) inserted strategically onto the capsid surface such that vector production and transduction efficiencies are minimally impacted. The LZ adaptors can then be used for modular and versatile attachment of proteins onto the capsid, such as cell-targeting nanobodies or peptides as well as genome editing reagents. Our central hypothesis is that Velcro AAVs will provide improved cell-targeting specificity and increased packaging capacity without affecting transduction efficiency, enabling safer and more robust somatic genome editing in vivo. During Phase 1 (UG3), Velcro AAV vectors will be constructed, characterized and optimized for nanobody-based endothelium-targeting (Aim 1a) and Cas9/BE protein attachment (Aim 2a). The effects of nanobody/nuclease attachment on viral titers and transduction efficiency will be quantified. Mouse studies will be carried out in Aims 1b, 2b and 2c to test the ability of Velcro AAV vectors to specifically target the endothelium with increased packaging capacity for gene editing in vivo. The targeting specificity and gene editing efficiency of Velcro AAV vectors will be further determined in Phase 2 (UH3) through pig studies in Aim 3. If successful, the proposed studies will yield strong preclinical demonstration of a new delivery platform technology that can provide specific cell/tissue targeting, larger cargo capacity, and transient nuclease activity, enabling safe and efficient somatic genome editing in humans.